1. Field of the Invention
This invention relates to bioremediation of contaminants in the environmental samples, including for example, contamination in particulates such as soil and also in fluids such as groundwater. More particularly, the present invention is directed to methods and compositions for the detection of perchlorate-reducing bacteria at target decontamination site.
2. Background of the Related Art
Chemical contamination of the environment, particularly of soil and groundwater, is a widespread problem throughout the industrialized world. Industrial pollution has contaminated millions of acres of soil and associated aquifers. Often, cleanup of the contamination is hindered because the cost of remediation is significant. Moreover, many of the remediation techniques create additional problems which cause the land to remain unused or abandoned.
Recently, widespread perchlorate contamination of drinking water wells throughout the United States and especially throughout the southwest has become a significant cause for concern. Perchlorate contamination of ground and surface waters originates from, and is a direct effect of, unregulated ammonium perchlorate disposal practices from 1950 to 1997 (Renner et al., Environ. Sci. Technol. News 32:210A, 1998). Ammonium perchlorate is an oxidant that is widely used in the aerospace, munitions, and fireworks industries. Widespread contamination has been documented in the waterways of California, and at least 19 other states in the United States. Similar contaminations have been reported in other countries that have aerospace, munitions and fireworks industries.
Perchlorate has been linked to a number of problems in human health. Excessive intake of perchlorate blocks iodine uptake and inhibits thyroid function and production of thyroid hormones, in addition, gastrointestinal irritation and skin rash, and hematological effects including agranulocytosis and lymphadenopathy have also been observed. In addition, it has been established that there is neurodevelopmental toxicity associated with perchlorate ingestion. As a result of these significant health concerns, drinking water utilities have begun monitoring and reporting perchlorate levels to the State agencies. In some states the Health Services Departments have set maximum limits on the amount of perchlorate in drinking water; this figure is typically in the order of 18 parts per billion (ppb) in order to minimize the risks to human health. The Environmental Protection Agency has established a provisional reference dose (“RFD”) of 14 mg of perchlorate per kg of body weight per day. Practical and efficient methods to treat water contaminated by perchlorate are needed to insure a safe drinking water supply in many communities.
Current methods of perchlorate remediation rely on the use of ion exchange resins to sequester perchlorate ions. (see e.g., U.S. Pat. No. 6,059,975). Conventional perchlorate-removal ion exchange resins have low selectivity coefficients and as such these resins are capable of loading only a few kilograms of perchlorate per cubic meter of removal resin. This produces a waste mass of loaded resin that must be disposed through, e.g., incineration. Disposal costs for these resins are therefore prohibitive because of the bulk volume of loaded to be disposed relative to the amount of perchlorate removed from the environmental target site. Other methods of perchlorate removal are actively being pursued, with bioremediation technologies emerging as a cost-effective and less-invasive alternative to physical or chemical practices (Urbansky et al., Biorem. J.: 81-95, 1998).
Natural attenuation of perchlorate is a cost-effective alternative to current methods of perchlorate remediation. Such natural attenuation systems have been used in bioremediation of other contaminants and include the use of microbial populations to accelerate the breakdown of solids and the various contaminants associated with waste water. Such microbes are permitted to act upon the waste water or contaminated soils and they act to remove the pollutants faster than if nothing were used, and do so without the hazards and difficulties associated with chemical treatment.
The success of natural perchlorate remediation is dependent on the presence and activity of dissimilatory (per)chlorate-reducing bacteria (DPRB) within the target site that is undergoing remediation. Within the last 7 years, more than 40 different strains of dissimilatory (per)chlorate-reducing bacteria (DPRB) have been isolated from a diverse range of environments (Bruce et al., Environ. Microbiol. 1:319-329, 1999; Coates et al., Appl. Environ. Microbiol. 65:5234-5241., 1999; Kim et al., Water Res. 35:3071-3076., 2001; Logan et al., Appl. Environ. Microbiol. 67:2499-2506, 2001, 18, Rikken et al., Appl. Microbiol. Biotechnol., 45:420-426, 1996; Wallace et al., J. Ind. Microbiol. 16:68-72, 1996). Because of the metabolic capability and ubiquity of DPRB (Coates et al., Appl. Environ. Microbiol. 65:5234-5241., 1999), natural attenuation of perchlorate is garnering more and more interest. While studies by various groups have shown the ability of microbes to remediate perchlorate under environmental conditions (Hunter, Curr. Microbiol. 45:287-292., 2002; Kim et al., Water Res. 35:3071-3076., 2001; Tipton et al., J. Environ. Qual. 32:40-46, 2003), a quick, reliable method for detecting the presence and effectiveness of DPRB is needed to determine the natural attenuation candidacy of a contaminated site as well as for monitoring active degradation.
Traditionally, contaminant site evaluation for the presence of DPRB has been performed using labor-intensive enumeration and isolation techniques. However, it is well known that cultivation techniques are time-consuming and often prove unsuccessful in isolating the target bacteria due to both media selectivity and organism culturability (Dunbar et al., Appl. Environ. Microbiol. 63:1326-1331, 1997; Kaeberlein et al., Science, 296:1127-1129, 2002). To alleviate the limitations of cultivation-based methods, molecular techniques using the 16S rRNA gene have been employed to examine bacterial diversity in the environment (Aman et al., Microbiol. Rev. 59:143-169, 1995, Olsen et al., Annu. Rev. Microbiol. 40:337-365, 1986), and numerous primer sets have been developed for the 16S rRNA gene that target specific groups of bacteria. However, due to the fact that there is significant phylogenetic diversity of DPRB and because of their close phylogenetic relationships to non-(per)chlorate-reducing relatives, detection of DPRB using 16S ribosomal DNA (rDNA) primers is not recommended (Achenbach et al., Int. J. Syst. Evol. Microbiol. 51:527-533, 2001). As such, there remains a need to identify a more inclusive approach to the detection of DPRB that would allow an efficient molecular identification technique that will facilitate the rapid identification of the presence of DPRB at a given target site and/or allow prediction of whether a given target site is capable of undergoing perchlorate remediation.